2009_Book_FoodChemistry

108 2 Enzymes
with a number of proteinases and esterases
alkylates the unusually reactive serine residue
at the active site. Thus, of the 28 serine residues
present in chymotrypsin, only Ser 195 is alkylated,
while the other 27 residues are unaffected
by the reagent. It appears that the reactivity
of Ser 195 is enhanced by its interaction with the
neighboring His 57 (cf. mechanism of catalysis
in Fig. 2.17). The participation of a carboxyl
group at the active site in β-glucosidase catalysis
has been confirmed with the help of conduritol
B-epoxide, an inhibiting substrate analogue:
(2.22)
A lysine residue is involved in enzyme catalysis
in a number of lyase enzymes and in enzymes
in which pyridoxal phosphate is the cosubstrate.
An intermediary Schiff base product is formed
between an ε-amino group of the enzyme and
the substrate or pyridoxal phosphate (cf. 2.3.2.3).
The reaction site is then identified by reduction
of the Schiff base with NaBH 4 .
An example of a “lysine” lyase is the aldolase enzyme
isolated from rabbit muscle. The intermediary
product formed with dihydroxyacetone phosphate
(cf. mechanism in Fig. 2.19) is detected as
follows:
(2.23)
2.4.1.2 Substrate Binding
2.4.1.2.1 Stereospecificity
Enzymes react stereospecifically. Before being
bound to the binding locus, the substrates are
distinguished by their cis, trans-isomerism and
also by their optical antipodes. The latter property
was illustrated by the reactions of L(+)-lactic
acid (Fig. 2.2). There are distinct recognition areas
on the binding locus. Alcohol dehydrogenase
will be used to demonstrate this. This enzyme removes
two hydrogen atoms, one from the methylene
group and the other from the hydroxyl group,
to produce acetaldehyde. However, the enzyme
recognizes the difference between the two methylene
hydrogens since it always stereospecifically
removes the same hydrogen atom. For example,
yeast alcohol dehydrogenase always removes the
pro-R-hydrogen from the C-1 position of a stereospecifically
deuterated substrate and transfers
it to the C-4 position of the nicotinamide ring of
NAD:
(2.24)
To explain the stereospecificity, it has been
assumed that the enzyme must bind simultaneously
to more than one point of the molecule.
Thus, when two substituents (e. g. the methyl
and hydroxyl groups of ethanol; Fig. 2.9) of
the prochiral site are attached to the enzyme
surface at positions A and B, the position of the
third substituent is fixed. Therefore, the same
substituent will always be bound to reactive
position C, e. g. one of the two methylene
hydrogens in ethanol. In other words, the two
equal substituents in a symmetrical molecule
are differentiated by asymmetric binding to the
enzyme.